Methods of fermentation can be largely classified into Batch fermentation and Fed-Batch fermentation, and continuous fermentation. Batch fermentation and Fed-Batch fermentation can be carried out with a simple equipment and finished in a short time, and is therefore less prone to be suffered from contamination, which is advantageous. However, there is a problem in that, as the product concentration in the culture increases with time, the productivity and the yield decrease due to the influence(s) of the osmotic pressure, product inhibition and/or the like. Therefore, it is difficult to maintain the stability, a high yield and a high productivity for a long time. Continuous fermentation has an advantage in that accumulation of a desired substance in the fermenter at a high concentration can be avoided and thereby a high yield and a high productivity can be maintained for a long time, but it is very difficult to stably continue culture by continuous fermentation for a long time, so that researches have been carried out therefor.
As a proposal for continuous fermentation, there is a method wherein microorganisms or cultured cells are filtered through a separation membrane and the product is recovered from the filtrate, while the microorganisms or cultured cells which were subjected to the filtration are retained in or returned to the culture to maintain a high concentration of the microorganisms or cultured cells in the culture.
For example, technologies wherein continuous fermentation is carried out in a continuous fermentation device having a ceramic membrane are disclosed (JP 5-95778 A, JP 62-138184 A, JP 10-174594 A and JP 2005-333886 A). However, the disclosed technologies have problems of decreases in the filtration flow rate and the filtration efficiency due to clogging of the ceramic membrane, so that reverse washing or the like is carried out to prevent the clogging.
A method for producing succinic acid by continuous fermentation (JP 2007-252367 A) is also disclosed. In this technology, a high filtration pressure (about 200 kPa) is employed for membrane separation. Since a high filtration pressure is disadvantageous in terms of not only the cost but also physical damage to microorganisms or cells by the pressure during filtration, it is inappropriate for continuous fermentation wherein microorganisms or cells are continuously returned to the culture. In JP 2007-252367 A, as proposals for maintaining continuous fermentation for a long time, technologies for the separation membrane, the filtration pressure and the like are disclosed, but the length of time of the continuous fermentation is about 300 hours, and therefore a method to maintain continuous culture for a longer period of time is demanded.
On the other hand, studies on microorganisms to be used for producing organic acids have been intensively carried out employing yeasts having high tolerances for acids (Biotechnology Progress, 11, 294-298 (1995) and Journal of Fermentation and Bioengineering, 86, 284-289 (1998)). In yeasts, there are haploid yeasts having only single sets of chromosomes and polyploid yeasts having pluralities of sets of chromosomes. Polyploid yeasts are used mainly as baker's yeasts and brewer's yeasts (JP 2000-139326 A, JP 2002-027974 A and JP 2002-253212 A). These are used for production of foods and beverages for the purposes of enhancement of flavor and improvement of the production process, and there is no description of their usage for continuous culture.
Further, methods for producing lactic acid using polyploids are disclosed (JP 2006-006271 A, JP 2006-020602 A, JP 2007-089466 A and JP 2001-204464 A). These employ polyploids to increase the number of the lactic acid synthetic gene, but the culture is carried out by Fed-Batch fermentation and the culturing time is as short as not more than 100 hours, and there is no description of continuous fermentation using a membrane.
In addition, production of lactic acid using prototrophic yeast which does not show auxotrophy was reported (US 2005/0112737 A), but also in this case, the culturing time is less than 100 hours, and Fed-Batch fermentation is the only method disclosed as a fermentation method.
Thus, continuous fermentation to enhance the productivity, and the microorganism to be used for fermentation have been separately studied. In cases where microorganisms were cultured by continuous fermentation which is advantageous as a fermentation method, the filtration pressure increased during the culture and continuation of the culture for a long time became impossible or the productivity of lactic acid decreased as the culture was continued longer, so that it has so far been difficult to allow exertion of the both advantages sufficiently. Thus, development of a technology for continuous fermentation which solves these problems and allows stable production of lactic acid for a long time has been demanded.
It could therefore be helpful to provide a method for production of lactic acid by continuous fermentation, by which a high productivity of lactic acid can be stably maintained for a long time.